The cytokine TNF, the pathogen recognition Toll-like receptors (TLRs) and the nucleotide binding oligomerization domain (NOD) proteins mediate host defense against infection in part by activating the transcription factor NF-icB. Our published work reveals Rip1 as a critical mediator of the TNF- and the TLR3/4, Trif-dependent NF-icB pathways. We have shown that the kinase activity of Rip1 is not required, but rather the ubiquitin modification of Rip1 is essential for TNF-induced NF-KB activation and cytokine production. In addition to mediating the Trif pathway, Rip1 has been implicated in dsRNA recognition by the cytosolic Rig-l/Mda5 helicases, however, it remains to be proven how a Rip1 deficiency impairs innate antiviral responses. To address this question, we will isolate Rip1-deficient macrophages and dendritic cells from hematopoeitic chimeras and conditional ripl mice and will infect these cell types with viruses and examine anti-viral signaling (Aim 1). Our preliminary studies reveal a novel regulatory role for Rip1 in the ubiquitination and activation of the interferon regulatory factor 7 (IRF-7), a transcription factor critical for type interferon production. Yet, how Rip1 regulates IRF-7 activity is unclear and a focus of the current proposal (Aim 1). An additional goal is to determine whether polyubiquitinated Rip1 is required for the activation of NF-xB and/or IRF-7 in virally infected cells and to test whether an inability to ubiquitin modify Rip1 results in impaired innate anti-viral responses (Aim 2). Similarly, our preliminary studies on the related Rip1 protein, Rip2, find endogenous Rip2 polyubiquitinated in cells treated with the NOD2 ligand, MDP and implicate the ubiquitin activated kinase Tak1 and the E2 ubiquitin conjugating enzyme Ubc13 in NOD2-mediated NF-icB activation. In Aim 3, we will test a requirement for polyubiquitinated Rip2 in NOD2-mediated NF-KB activation and in innate immune responses and will determine how disease associated alleles of NOD2 affect the recruitment and polyubiquitination of Rip2. Collectively, our studies suggest that ubiquitin regulates innate immune responses, raising the possibility that the enzymes responsible for the ubiquitin modification of Rip proteins may be targeted therapeutically to treat infectious disease or chronic inflammatory disease. Bacteria and viruses are recognized by receptors designed to fight infection. These receptors respond by producing soluble factors that have anti-bacterial and anti-viral activity. Our research is focused on how Rip proteins contribute to host responses against infection with the goal that the activity of Rip proteins can be stimulated or attenuated by drugs as needed, in human disease.